1. Field of the Invention
This invention relates to a method for determination of DNA and a sensor therefor. More particularly, it provides a method for detection of DNA which permits the detection to be performed expeditiously with a simple procedure and also provides a sensor to be used for such determination.
2. Description of the Prior Art
(1) General methods of testing for DNA:
Prior to the present invention, various means for DNA testing and DNA diagnosis have been disclosed, including, e.g., dot blot methods, in situ hybridization methods, Southern blotting methods, PCR methods, and polygonal DNA marker methods, However, all of such methods have problems. For example, the dot blot method and the in situ hybridization method have problems of poor sensitivity of detection, although they are advantageous in the fact they are relatively simple and require little time for DNA detection. By contrast, Southern blotting methods provide high sensitivity of detection, but are complicated and time-consuming. Means of DNA analysis which obviates some methods associated with DNA testing and DNA diagnosis, are the Sanger methods and the Maxam methods of DNA detection. However, such methods are still disadvantageous because it is difficult to obtain ideal analysis of DNA which is composed of more than 1,000 base pairs.
F. Sanger et al have published in Proc. Natl. Acad. Sci., USA 74, 5463 (1977) a method for identifying a base sequence in a given DNA by using a reaction for extracting one of the four precursor nucleotides and a reaction for incorporating one precursor nucleotide therein.
A. Maxam and W. Gilbert have published in Proc. Natl. Acad. Sci., USA 74, 560 (1977) a method for sequencing a base sequence contained in a given DNA by labeling the 5'-terminal of the DNA with .sup.32 P, reacting said DNA with a chemical reagent, thereby inducing partial decomposition of the DNA to its bases, and the formation of DNA fragments differing in length, and fractionating the DNA fragments by electrophoresis. However, this method requires that the terminal labeled pure DNA be available in a large amount, therefore it requires amplification of the DNA by cloning, by PCR or by LCR. Thus, this method is time-consuming and costly.
The PCR method features unusually high sensitivity because it is adapted to effect propagation of the DNA at the target site to 100,000 to 1,000,000 times the original size. It, however, is subject to contamination and complicated operating conditions.
Conventional apparatuses for DNA testing are subject to various problems including consumption of an unduly long time, requirement of special test room conditions because of required use of radiation, and need for means of disposal of hazardous spent isotopes. Thus, there is a substantial need for the development of a DNA sensor which provides for high sensitivity but which does not require use of an isotope.
(2) QCM
Successful use of a quartz crystal microbalance (QCM) for DNA assay has been disclosed to the art. This device comprises a quartz plate and electrodes formed on each of the opposite surfaces of the quartz plate. These two electrodes are connected to external oscillating circuits and are adapted to resonate with the frequency inherent in the quartz plates. This frequency is related to the mass of quartz as well as the mass, viscosity, and viscoelasticity of the electrodes which are in contact with the quartz. Generally, the variation of resonant frequency and that of mass of a substance in contact with quartz are correlated. Assuming that the layer and/or substance in contact with quartz behaves as a rigid body, the variation of mass may be calculated in accordance with the Sauerbrey formula [G. Z. Sauerbrey, Z. Phys., 155 (1959) 206] shown below. EQU .DELTA.f=2f.sub.0.sup.2 .DELTA.m/A(.rho..sub.q .mu..sub.q).sup.1/2( 1)
wherein .DELTA.f is a shift of frequency, f.sub.0 is fundamental frequency of quartz, .DELTA.m is a variation of mass, A is a available surface area of an electrode, .rho..sub.q is a density of quartz (2.648 g cm.sup.-3), .mu..sub.q is a constant of shift vibration (which is 2.947.times.10.sup.11 dynes cm.sup.-2 in the case of AT cut crystal).
R. C. Ebersole and M. D. Ward have disclosed in U.S. Pat. No. 4,999,284 and in J. Amer. Chem. Soc., (1988), 110, 8623-8628 a method which provides for detection determination of an immune substance with high sensitivity which comprises immobilizing the immune substance on a quartz oscillator. These publications touch on DNA but fail to show a concrete procedure.
J. C. Andle et al. have reported in Sensors and Actuators B. 8 (1991) 191-198 the successful detection of DNA by the use of a sensor comprising a so-called SAW device having a comb electrode formed on the surface of a piezoelectric plate. In their report, the sensitivity of DNA detection is indicated to be 0.1 nanogram in mass sensitivity at a phase of 0.5.degree..
In J. Amer. Chem. Soc., (1992), 118, 8299-8300, Y. Okahata et al. have disclosed a method which allows for the detection of a variation of weight of a given oligonucleotide due to hybridization by immobilizing 10mer of a SH Group-modified sample of the oligonucleotide on the surface of a gold electrode of a quartz oscillator, exciting the quartz so as to induce oscillation, measuring the variation of resonance frequency generated by the oscillation, and calculating the magnitude of the variation of weight based on the variation of the resonant frequency.
These methods provide for both DNA detection and quantitative measurement of test DNA in a sample on the basis of the variation in resonance frequency. For practical use in DNA diagnosis and testing, however, the method must warrant sensitivity of not less than 10.sup.-16 mol. The methods cited above provide DNA detection sensitivity on the order of 0.1 to 1 nanogram. They, therefore, have a problem of impracticability due to unduly low sensitivity. Ideally, a method should enable detection of a single DNA. Thus, the development of a method and apparatus which allows for detection of a single DNA is needed. Even assuming a sample DNA to be detected is large, e.g. a DNA having a molecular weight of 10.sup.9 such as a human gene, it still cannot be detected using conventional methods because the mass per molecule is 1.7.times.10.sup.-15 (10.sup.9 /6.02.times.10.sup.23) g.
Moreover, the method proposed by Okahata et al. is not fully reliable because it must rely on aspiration for immobilization of DNA. Thus, it is subject to inaccurate determination of DNA.
An object of this invention, therefore is to provide a novel method for determination of DNA and a sensor to be used therefor.
Another object of this invention is to provide a DNA sensor which is formed of an elastic wave element which is so supersensitive that it allows for the immobilization of a modified DNA which enables detection of a single DNA and to provide a method for the determination of DNA using this DNA sensor.
Yet another object of this invention is to provide a base sequence reading apparatus which permits highly sensitive detection or isolation of DNA and which allows highly accurate detection of a base sequence in the DNA for DNA sequence analysis.